Ever since the discovery of penicillin in the 1920s, and streptomycin in the 1940s, many new compounds with antibacterial activity have been discovered and used as antibiotics, which have saved thousands of lives and greatly contributed to the development of modern animal husbandry industry. But over time, bacteria can become resistant to existing drugs, making infections difficult to treat or even impossible to control. Currently, antibiotic-resistant bacteria has become a growing public health threat, as exemplified by the long-existing Methicillin-resistant Staphylococcus aureus (MRSA) and the recent emergence of NDM-1 superbugs. In fact, almost all of the clinical used antibiotics have been found to eventually lead to the emergence of resistant bacteria strains, such as drug-resistant strains of Gram-positive bacteria, methicillin-resistant staphylococcus, streptococcus, penicillin-resistant and vancomycin-resistant enterococci, etc. When these resistant bacteria infect patients or animals they cause serious or even fatal consequences. In the field of drug research the development of new antibiotics is an important way to combat the problem of drug-resistant.
Macrolides are a class of 14-16 membered lactone antibiotics substituted with one or more deoxy sugars, including erythromycin, tylosin, tilmicosin, roxithromycin, erythromycin, azithromycin, clarithromycin, spiramycin, tulathromycin, oleandomycin, carbomycin, and flurithromycin, etc. Macrolides exert their bacteriostatic effect by binding reversibly to the P site on the subunit 50S of the bacterial ribosome, inhibiting bacterial protein synthesis through preventing peptidyltransferase from adding the growing peptide attached to tRNA to the next amino acid, as well as inhibiting ribosomal translation, similarly to the mechanism of action of chloramphenicol and lincosamides antibiotics. Another potential mechanism is premature dissociation of the peptidyl-tRNA from the ribosome.
Macrolide antibiotic resistant bacteria also have emerged. The mechanisms of resistance include: (1) by reducing the permeability of the bacterial cell wall or acquired efflux mechanism to reduce drug accumulation in cells; (2) by ermA, ermB and ermC gene mediated methylation of 50S ribosome binding sites, thereby greatly reducing the affinity of antimicrobial agents to the ribosome binding site; (3) enzymatic inactivation of the drug by the bacteria due to induced production of ester hydrolase.
Tylosin and its associated 16-membered macrolide derivatives (see FIG. 1) have already proven to be effective against certain infections caused by Gram-positive and Gram-negative bacteria in animals such as poultry, cattle, and pigs. (Kirst et. al., U.S. Pat. Nos. 4,468,511, 4,920,103; Tao et. al., U.S. Pat. No. 4,921,947; Lukacs et. al., U.S. Pat. No. 5,032,581). The chemical structures of Tylosin, Tilmicosin, and Tildipirosin are shown in FIG. 1. However, tylosin suffers relatively low bioavailability, gastrointestinal side effects, and limited spectrum of antibacterial activity. Tilmicosin is a 20-modified tylosin derivative with significantly improved pharmacokinetic properties, especially the longer half-life (U.S. Pat. No. 5,545,624). Its main disadvantage is cardiac toxicity, particularly when administered by injection.
Another tylosin related macrolide derivative Tildipirosin retained the piperidinyl base substructure of the tilmicosin at the 20-position but further modified the substituent at position 23, replacing the mycinose with another piperidinyl ring, resulted in higher activity against Mannheimia haemolytica and Pasteurella multocida, which are the two main etiological agents of bovine respiratory disease (U.S. Pat. No. 6,514,946 B1). Tildipirosin and Tulathromycin both contain three basic amino groups, which could contribute to the enrichment in the lung tissue and bronchoalveolar fluid and the longer half-life of these triamilides. However, while two identical piperidines at positions 20 and 23 may have improved the efficacy against certain pathogens and circumvented stereoisomeric issues of the 20-dimethylpiperidine in Tilmicosin, novel modifications at positions 20 and 23 could potentially provide agents of further improved antibacterial profile and reduced resistance. Stephen Douthwaite et al have recently studied the inhibition of protein synthesis on the bacterial ribosome by tylosin related macrolides and suggested that positions 20 and 23 of the macrolide molecules are closely associated with nucleotide A2058 and G748 for binding affinity, where mutation or methylation are responsible for the observed resistance (Antimicrob. Agents Chemother. 2012, 56(11):6033). It has also been suggested by a computer assisted modeling study that the interaction of 23-piperidine of Tildipirosin with the binding tunnel is slightly more distal from G748 (ACS Chem. Biol. 2012, 7, 1351-1355) and we believe that the protonation state of the piperidine nitrogen is also critical to the binding affinity.
A variety of structural modifications of tylosin macrolide have been reported over the years, for example, U.S. Pat. No. 4,468,511A, GB2135670A, U.S. Pat. No. 6,514,946, and references therein described 20- and 23-modified tylosin macrolides. More recently, Hong Fu et al described tylonolide 9- and 20-modified derivatives with ketolide-like activity against macrolide-resistant S. pneumoniae and inducible-resistant S. aureus strains in vitro. (Bioorganic & Medicinal Chemistry Letters 2006, 16, 1259-1266).